Methods in molecular biology
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In a normal spontaneous menstrual cycle, the luteal phase is characterized by the production and secretion of estradiol (E) and progesterone (P) from the corpus luteum (CL) in an episodic manner. The steroidogenesis of the CL is dependent on continued tonic luteinizing hormone (LH) secretion (Fritz and Speroff, Clinical gynecologic endocrinology and infertility, 8th edn. Wolters Kluwer, Lippincott Williams & Wilkins, Philadelphia, 2011). ⋯ Progesterone concentrations normally rise sharply after ovulation, reaching a peak approximately 8 days after the LH surge. Since the secretion of E and P during the luteal phase is episodic and correlates closely with LH pulses, relatively low mid-luteal progesterone levels can be found in the course of a totally normal luteal phase (Fritz and Speroff, Clinical gynecologic endocrinology and infertility, 8th edn. Wolters Kluwer, Lippincott Williams & Wilkins, Philadelphia, 2011).
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Targeted intrathecal (IT) drug delivery systems (IDDS) are an option in algorithms for the treatment of patients with moderate to severe chronic refractory pain when more conservative options fail. This therapy is well established and supported by several publications. ⋯ Recent technological advances, new therapeutic applications, reported complications, and the costs as well as maintenance required for this therapy require the need to stay up-to-date about new recommendations that may improve outcomes. This chapter reviews all technological issues regarding IDDS implantation with follow-up, and pharmacological recommendations published during recent years that provide evidence-based decision making process in the management of chronic pain and spasticity in patients.
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The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have evolved as an adaptive surveillance and defense mechanism in bacteria and archaea that uses short RNAs to direct degradation of foreign genetic elements. Here, we present our protocol for utilizing the S. pyogenes type II bacterial CRISPR system to achieve sequence-specific genome alterations in human cells. ⋯ Genomic alterations at the target site are then introduced either through nonhomologous end joining (NHEJ) or through homologous recombination (HR) in the presence of an appropriate donor sequence. This RNA-guided editing tool offers greater ease of customization and synthesis in comparison to existing sequence-specific endonucleases and promises to become a highly versatile and multiplexable human genome engineering platform.
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N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that are essential for synaptic plasticity, learning and memory. Dysfunction of NMDARs has been implicated in many nervous system disorders; therefore, pharmacological modulation of NMDAR activity has great therapeutic potential. However, given the broad physiological importance of NMDARs, modulating their activity often has detrimental side effects precluding pharmaceutical use of many NMDAR modulators. ⋯ Additionally, the effects of drugs on agonist-activated responses typically depend on duration of agonist exposure. To evaluate drug effects on synaptic transmission, an approach should be used that allows for activation of receptor responses as brief as those observed during synaptic transmission, both in the absence and presence of drug. To address these issues, we designed a fast perfusion system capable of (1) delivering brief (~5 ms) and consistent applications of glutamate to recombinant NMDARs of known subunit composition, and (2) easily and quickly (~5 s) changing between glutamate applications in the absence and presence of drug.
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Genome sequencing and systems biology are revolutionizing life sciences. Proteomics emerged as a fundamental technique of this novel research area as it is the basis for gene function analysis and modeling of dynamic protein networks. Here a complete proteomics platform suited for functional genomics and systems biology is presented. ⋯ Moreover, the presented platform can also be utilized to integrate metabolomics and transcriptomics data for the analysis of metabolite-protein-transcript correlations and time course analysis using COVAIN. Examples for the integration of MAPA and MASS WESTERN data, proteogenomic and metabolic modeling approaches for functional genomics, phosphoproteomics by integration of MOAC (metal-oxide affinity chromatography) with MAPA, and the integration of metabolomics, transcriptomics, proteomics, and physiological data using this platform are presented. All software and step-by-step tutorials for data processing and data mining can be downloaded from http://www.univie.ac.at/mosys/software.html.